Polyethylene glycols are a family of polymers produced from the condensation of ethylene glycol, and have the general formula H(OCH.sub.2 CH.sub.2).sub.n OH where n, the number of ethylene glycol groups, is greater than or equal to 4. Generally, the designation of a polyethylene glycol (PEG) includes a number that corresponds to its average molecular weight. For example, polyethylene glycol 1500 refers to a mixture of polyethylene glycols having an average value of n between 29 and 36 and a molecular weight range of 1300 to 1600 grams/mole.
The properties of polyethylene glycols vary with the polymer's molecular weight. Polyethylene glycols have been used in plasticizers, softeners and humectants, ointments, polishes, paper coating, mold lubricants, bases for cosmetics and pharmaceuticals, solvents, binders, metal and rubber processing, permissible additives to foods and animal feed, and laboratory reagents, among others.
Polyethylene glycols are generally linear or branched, neutral polyether molecules that are soluble in water and organic solvents. In addition to the uses noted above, polyethylene glycols have proven to be valuable in many biotechnical and biomedical applications. Polyethylene glycols have been advantageously employed in these applications for their ability to impart water solubilization and surface protective properties, and also because these polymers are only weakly immunogenic.
Polyethylene glycols have also been covalently coupled to proteins to alter their properties in ways that extend their potential uses. Due to in vivo instability, the efficacy of a number of therapeutic proteins is severely limited. While many approaches to stabilization of such proteins have been made, the covalent modification of proteins with hydrophilic polymers such as dextran and polyethylene glycols has been most successful. Typically, polyethylene glycol-protein conjugates are more stable than the native protein in vivo and often, the modified proteins exhibit enhanced resistance to proteolytic degradation. The result is an increase in the therapeutic protein's life in circulation and a reduction in its immunogenicity. In some instances, the therapeutic efficiency of these conjugates is greatly enhanced compared to the native protein.
The improved performance of PEG-modified conjugates has resulted in their development as therapeutic agents. For example, enzyme deficiencies where the native enzyme was ineffective due to its rapid clearance and/or immunological reactions have now been treated with equivalent PEG-enzymes. In fact, PEG-adenosine deaminase has obtained FDA approval. Because PEG-enzymes can act as catalysts in organic solvents, the use of such PEG-enzymes to produce desired stereoisomers offers an advantage to classical organic synthesis which typically produces racemic mixtures of organic products. See, e.g., Degado et al., Crit. Rev. Ther. Drug Carrier Systems, Vol. 9, pp. 249-304, 1992.
Examples of polyethylene glycol-modified proteins include PEG-adenosine deaminase (PEG-ADA), which has been used in enzyme replacement therapy for immunodeficiency due to ADA deficiency (M. S. Hershfield, Clin. Immunol. Immuno. Pathol., Vol. 76, S 228-232, 1995); PEG-recombinant human granulocyte colony stimulating factor (PEG-rhG-CSF), which showed an increase in stability and retention of in vivo bioactivity and has been suggested as a suitable form of the protein for inclusion in an oral delivery formulation (P.-K. E. Jensen et al., Pharm. Res., Vol. 13, pp. 102-107, 1996); PEG-natural human tumor necrosis factor alpha, which showed a gradual decrease in specific activity with increasing degree of PEG-modification and a drastic increase in plasma half-life upon PEG-modification (Y. Tsutsumi et al., Br. J Cancer, Volume 71, pp. 963-968, 1995); PEG-recombinant human interleukin-2, which retains the in vitro and in vivo activity of interleukin-2, but exhibits a markedly prolonged circulating half-life (T. Menzel et al., Cancer Bio. Ther., Vol. 8, pp. 199-212, 1993); and PEG-asparaginase, which has shown promise in patients suffering from acute lymphocytic leukemia (N. Burnham, Am. J Hosp. Pharm., Vol. 52, pp. 210-218, 1994). Polyethylene glycol conjugates of oligonucleotides have also been prepared and show a more than tenfold increase in exonuclease stability (A. Jaschke et al., Nucleic Acids Research, Vol. 22, pp. 4810-4817, 1994).
Other PEG-modified proteins include papain (C. Woghiren et al., Bioconjugate Chemistry, Vol. 4, pp. 314-318, 1993), asialofetuin (L. Roseng et al., J. Biol. Chem., Vol. 267, pp. 22987-22993, 1992), collagen (C. J. Doillon et al., Biomaterial Sciences Polymers, Vol. 6, pp. 715-728, 1994), RGDT peptides (I. Saiki, Japanese J. Cancer Research, Vol. 84, pp. 558-565, 1993), serum IgG (R. Cunningham et al., J. Immunol. Methods, Vol. 152, pp. 177-190, 1992), alpha 1-proteinase inhibitor (A. Mast et al., J. Lab. Clin. Med, Vol. 116, pp. 58-65, 1990), growth hormone releasing factor (A. Felix, Int. J. Peptide Protein Research, Vol. 46, pp. 253-264, 1995), basic fibroblast growth factor (S. Kusstatscher et al., J. Pharmacol. Exp. Ther., Vol. 275, pp. 456-61, 1995), and catalase, uricase, honey bee venom, hemoglobin, and ragweed pollen extract. As indicated by the number of utilities noted above, polyethylene glycol has recently been widely used to develop new therapeutic agents.
Despite the widespread use of polyethylene glycols to modify therapeutic agents, their use has not been without associated disadvantages. The covalent attachment of polyethylene glycol to superoxide dismutase produces a heterogeneous mixture of modified protein species. The heterogeneity of the product derives from, in part, the polydispersity of the polyethylene glycol reagent (J. Snyder et al., J. Chromatography, Vol. 599, pp. 141-155, 1992.)
Commercially available polyethylene glycols having molecular weights greater than about 300 grams/mole are available only as mixtures of varying length polymers. The range of PEG polymer lengths results from the polymerization process by which the PEG polymers are prepared. Commercially available PEG polymers include polymers having average molecular weights of 100, 200, 300, 400, 600, 900, 1000, 1500, 2000, 3400, 4600, 8000, and 10000 grams/mole from Aldrich Chemical Co. (Milwaukee, Wis.); 3350 and 20000 grams/mole from Sigma Chemical Co. (St. Louis, Mo.); and 1000, 2000, 3000, 5000, 10000, 20000 and 25000 grams/mole from Shearwater Polymers, Inc. (Huntsville, Ala.). The exact composition of these mixtures are not generally provided, but are considered of relatively narrow range except where terminal monomethyl ethers are desired.
Inherent problems with the utilization of such polymeric mixtures of PEG molecules exist. One of the most significant problems is that a mixture of compounds is obtained when these compounds are modified or used to modify other compounds. Having a mixture of compounds complicates purification and characterization of the compounds. Further, even though PEG molecules are relatively innocuous in the biological system, different compounds within these mixtures are likely to have different pharmacokinetics, pharmacodynamics, and even varying degrees of toxicity, making such a mixture questionable for pharmaceutical applications.
Yet another consideration in having mixtures is the role of sizes in obtaining the desired biological properties. While small differences might be expected in solubilization within a "narrow" average molecular weight range, properties such as rendering proteins nonimmunogenic may be compromised by having a mixture of sizes on the protein surface. The fact that there are variances in lengths may make the protein more recognizable. While immunogenic proteins modified by PEG compounds often have benefits arising from that process, the fact that a mixture of PEG compounds is used may result in obtaining weakly immunogenic responses.
Accordingly, there remains a need in the art for alternatives to PEG polymers composed of a mixture of lengths and molecular weights to overcome the difficulties associated with the preparation, purification, characterization, and therapeutic administration of such PEG mixtures. The present invention seeks to fulfill these needs and provides further related advantages.